Compounds of hydrophilic polymer-polycarboxyl oligopeptide and medicines, medical composite comprising above compound and use of above compound in medicimes

ABSTRACT

The present invention relates to a conjugate of hydrophilic polymer-multicarboxyl oligopeptide and drug molecule of the following formula:  
                 
 
wherein: P is a water soluble polymer; m is an integer of 2˜12; j is an integer of 1˜6; R i  is a group selected from H, C 1-12  alkyl, substituted aryl, aralkyl, heteroalkyl and substituted alkyl; X and Z are linking groups; and TA is drug molecule. The conjugate has low toxicity and an ability to carry more than one drug molecule to improve solubility, sustain and control drug release, and has a remarkably enhancing effect especially to antitumor drug such as paclitaxel and camptothecin etc.

FIELDS OF THE INVENTION

The present invention relates to a new conjugate of hydrophilicpolymer-multicarboxyl oligopeptide and drug molecule, a pharmaceuticalcomposition comprising the conjugate and the pharmaceutical use of theconjugate.

BACKGROUND OF THE INVENTION

Among the active constituents of nature medicine, proteins,polypeptides, terpenoids, steroids, alkaloids, flavonoids,anthraquinones, and phenylpropanoid phenols all show various effectiveproperties in the term of biologically activity, and therefore, they'vebeen widely used in medicine. Their glycoside, nucleoside andpolypeptide derivatives have also shown considerable applications. Asnatural active constituents, they have the advantages of fastbiodegradation rate, little or no residue, low toxicity, and little orno side effects. However, they still have some disadvantages such as lowbioavailability, short physiological half-life, poor water solubility,immunogenecity and the like.

To solve the problems, the derivatives of PEG have been widely used toconjugate with proteins, peptides or other therapeutic agents in orderto prolong their physiological half-life and to lower theirimmunogenicity and toxicity. Clinically, PEG and its derivatives havebeen widely used as carrier to manufacture preparations of commercialdrugs. And the attempt of conjugating PEG to drug molecule has animpressive progress in the last 10 years and has been applied to manyofficially approved drugs. For example, PEG-intron®, a conjugate of PEGand α-interferon, exhibits longer circulation half-life and bettertherapeutic effect compared to the native form of α-interferon. It hasbeen shown that the conjugate of PEG and paclitaxel correspondinglyreduces the toxicity and prolongs the bioactivity. The metabolismprocesses of these conjugate are well known, showing that PEG is a safedrug modifier.

When conjugating PEG to drugs, often is used a process called asPEGylation, in which one or two of the terminal groups of PEG arechemically activated to have a proper functional group which is reactiveto at least one functional group of the drug to form a stable bond. Thisstable bond can be eliminated by degradation under proper conditions invivo, and thereby the active ingredient is released.

It's reported that PEG can be used to conjugate to many drugs. Theprodrugs of PEG derivatives conjugating with paclitaxel have beendisclosed in U.S. Pat. Nos. 5,824,701 and 5,840,900 and CN patentCN1283643. In these patents, each of the two terminal groups of PEG isconjugated with a paclitaxel molecule. In order to increase the drugmolecule load, U.S. Pat. No. 6,153,655 discloses a terminally branchedPEG structure, in which two functional groups are formed via aminolinkage at the two termini of PEG. However, the introduction ofnon-biological branched small molecule also makes the drug propertyindefinite. U.S. Pat. Nos. 5,977,163 and 6,262,107 and Chinese patentCN1164533 disclose a paclitaxel prodrug with polyglutamic acid ascarrier, in which paclitaxel is attached randomly to the active carboxylgroups of glutamic acid along the polyglutamic acid skeleton. The broadpolydispersity and uncertainty of the toxicity of polyglutamic acidlimit the application of these inventions.

SUMMARY OF THE INVENTION

Therefore, the object of the present invention is to provide a conjugateof the hydrophilic polymer-multicarboxyl oligopeptide and drug moleculerepresented by the following formula:

wherein:

-   P is a water-soluble polymer;-   m is an integer from 2 to 12;-   j is an integer from 1 to 6;-   R_(i) is a group selected from the group consisting of H, C₁₋₁₂    alkyl, substituted aryl, aralkyl, heteroalkyl and substituted alkyl;-   X is a linking group;-   Z is a linking group selected from O and NH; and-   TA is a drug molecule.

According to another aspect of the invention, there is provided apharmaceutical composition comprising the above conjugate as activeingredient.

According to still another aspect of the invention, there is provided ause of the above conjugate in preparing a pharmaceutical composition.

The conjugate of the present invention can improve the absorption ofdrugs, prolong therapeutic duration, increase therapeutic effect, reducedosage, and avoid toxicity and other side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a more detailed description of the present invention byreferring to the following drawings, in which:

FIG. 1 shows the synthesis of PEG-glutamic acid oligopeptidederivatives;

FIG. 2 shows the synthesis of ester bonding conjugates of PEG-glutamicacid oligopeptide derivatives and drugs; and

FIG. 3 shows the synthesis of other kind of conjugates of PEG-glutamicacid oligopeptide derivatives and drugs.

DETAILED DESCRIPTION OF THE INVENTION

The hydrophilic polymer used in the conjugate of the present inventionis, for example, polyethylene glycol, polypropylene, polyvinyl alcohol,polyacrylmorpholine or copolymer thereof, among them, polyethyleneglycol and its copolymer are preferable. Acidic oligopeptide of aminoacid, especially oligopeptide of glutamic acid is conjugated to theparent polymer by modifying free end hydroxyl of such hydrophilicpolymer. This conjugation provides linking sites between the polymer anddrug molecule. In this way, free amino and hydroxyl of proteins,peptides or other the active ingredients of nature medicine can belinked to the polymer. Especially for the active ingredient of smallmolecule nature medicine, one or more drug molecules can be linked tothe hydrophilic polymer-multicarboxyl oligopeptide by this way in orderto ensure an appropriate drug concentration and sustained release.

In the conjugate according to the present invention, multicarboxylgroups of glutamic acid oligopeptide provide many conjugation sites, andthe conjugate thus has a higher drug load than the usual linear PEGcarrier. The difference between glutamic acid oligopeptide andpolyglutamic acid is that glutamic acid oligopeptide has a definitenumbers of active carboxyl groups per oligopeptide chain. Therefore,when conjugating with drug molecule such as paclitaxel, it will be easyto confirm and repeat the loading amount of the drug. At the same time,because of non-hydrophilicity of the drugs such as paclitaxel, theconjugate of hydrophilic polymer-glutamic acid oligopeptide-drugmolecule will form a molecular micelle/microsphere structure consistingof a number of aggregating molecules in aqueous solution. This structurewill maintain the favorable properties of the hydrophilic polymer, suchas hydrophilicity, flexibility, and anti-macrophage phagocytosis.Meanwhile, the structure provides sustained release of the drug moleculeand prolonged retention time in vivo for the drug, especially naturemedicines.

One of the advantages of the present invention is that, in addition ofthe characteristics of the hydrophilic polymer such as PEG or itsderivatives, for example, solubility, non-immunogenicity andnon-toxicity, the oligopeptide groups provide many loading sites for thedrug molecules, to ensure the effective blood concentration and thestepwise release of the drug.

Now we'll take PEG derivatives as an example to explain the linkagebetween hydrophilic polymer and multicarboxyl oligopeptides.

The structure of PEG derivatives includes the polymeric branched chainpart and the terminal functional group part, which are describedrespectively as follows.

Polyethylene glycols (PEGs) are represented by the following generalformula below:

wherein:

-   R is H or C₁₋₁₂ alkyl; and-   n is an integer, representing the degree of polymerization.

As a lower alkyl, R can be any lower alkyl group having 1-6 carbonatoms, for examples, methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, n-pentyl, and n-hexyl. As a cycloalkyl, R is preferably acycloalkyl containing 3-7 carbon atoms, for example, cyclopropyl,cyclobutyl, and cyclohexyl. Among those, cyclohexyl is more preferable.The typical compound is methoxy-polyethylene glycol (mPEG). Otheranalogs and derivatives of polyethylene glycol, such as polypropyleneglycols, polyvinyl alcohols, and polyacrylmorpholines and the like, canalso be used in the present invention.

In respect of PEGs, they are usually measured by molecular weight. It'spreferred the molecular weight of PEG which forms the conjugate falls inthe range from 300 to 60000 Daltons, which means n is about 6˜1300. It'smore preferred that n is 28, 112 and 450, respectively corresponding tomolecular weight of 1325, 5000, and 20000. Because of the potentialnon-homogeneity of the starting PEGs which are usually defined by theirmolecular weights rather than the self-repeating unit n, PEGs arenormally characterized with average weight molecular weight, insteadthat PEGs are characterized with their slelf-repeating units representedby n. The starting PEG compounds with different molecular weights arereadily synthesized using the known methods of the art or arecommercially available.

The multicarboxyl oligopeptides are represented by the following generalformula:

wherein:

-   m is an integer of 2˜12, representing the degree of polymerization;-   j is an integer of 1˜6; and-   R_(i) is a group selected from the group consisting of H, C₁₋₁₂    alkyl, substituted aryl, aralkyl, heteroalkyl and substituted alkyl.

As a lower alkyl, R_(i) can be any lower alkyl group having 1-12 carbonatoms, for example, methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, n-pentyl and n-hexyl, and possible cycloalkyl. As an aralkyl,R_(i) is preferably benzyl or phenylethyl. The preferred substitutedaryl is benzyl.

The synthesis of oligopeptide may be carried out according to thegeneral synthesis method. With protective amino acid, amino acid polymercan be produced with high yield under the effect of dehydrant. Ofcourse, because the oligopeptide of the present invention is ahomo-polypeptide, there's no structural sequence problem here.Therefore, easier methods can be used herein, such as mixed acidanhydrides method, active ester method and N-carboxy-a-amino acidanhydrides (NCA) method etc.

Taking N-carboxy-a-amino acid anhydrides method as an example, thesynthesis is shown as follows:

The reaction has the advantages of fast reaction speed and shortsynthesis cycle. When the reaction is complete, the obtained peptideswith free amino group can be used directly in the next synthesis step ofoligopeptides without separation. The side chain generally needs lessprotection. Side chain protection is usually necessary for the sidechain of NCA. For amino component, only the side chains of lysine andcysteine need to be protected.

Multicarboxyl-oligopeptide compound are readily synthesized using knownmethods in the art or are commercially available.

The hydrophilic polymer-glutamic acid oligopeptide moiety of the presentinvention is synthesized from hydrophilic polymer and glutamic acidoligopeptide by the known methods of the art.

In practical application, the terminal groups of the hydrophilic polymerneed to be activated to ensure that it is capable of reacting with aminoor carboxyl groups of the oligopeptide to form the conjugated. Inrespect of the intended use, the terminal functional groups can bemodified with the following methods:

-   a. Amination. The amino that is of greater reactivity takes the    place of hydroxyl after the hydrophilic polymer is aminated. It's    especially important when the polymer reacts with a molecule    comprising carboxylic acid group to yield a conjugate.-   b. Carboxylation. Carboxylating the hydrophilic polymer improves its    reactivity, and makes it capable of conjugating to molecules    containing amino or carboxyl groups.-   c. Other methods such as modifications by acyl chloride, hydrazine,    maleimide, pyridine disulfide etc. can all be appropriately adopted    as well.

All of the methods listed above will lead to the formation of chemicalbonds between the functional groups of hydrophilic polymer andoligopeptide. Thus it's possible to make good use of the advantages ofthe two compounds.

All of the drugs used at present, especially nature medicines, comprisefunctional groups such as amino, carboxyl and hydroxyl groups. In vivo,these functional groups can conjugate with monosaccharide,polysaccharide, nucleoside, polynucleoside, and phosphoryl etc to formstructure pharmacologically active in vivo.

Consequently, hydrophilic polymer-multioligopeptide can conjugate to thedrug molecules in the same way to replace bio-organic molecules andovercome their shortcomings of short physiological half-time and shorttherapeutic duration. The hydrophilic polymer-multicarboxy oligopeptideof the present invention has the following formula:

wherein:

-   P is a water soluble polymer, which may be polyethylene glycol,    polypropylene glycol, polyvinyl alcohol, polyacrylmorpholine or    their copolymer, and polyethylene glycol and its copolymer are    preferred;-   m is an integer of 2˜12;-   j is an integer of 1˜6;-   R_(i) is a group selected from the group consisting of H, C₁₋₁₂    alkyl, substituted aryl, aralkyl, heteroalkyl and substituted alkyl;-   X is a linking group, preferably (CH₂)_(i), (CH₂)_(i)OCO,    (CH₂)_(i)NHCO and (CH₂)_(i)CO, wherein i is an integer of 1˜10;-   Z is a linking group selected from O and NH; and-   TA is a drug molecule.

If the hydrophilic polymer comprises free hydroxyl groups, the hydroxylgroups can be blocked by C₁₋₁₂ alkoxyl, cycloalkoxyl or aroxyl,preferably by methoxyl, ethoxyl, iso-propoxyl, cyclopropoxyl,cyclobutoxyl, cyclohexoxyl or benzoxyl.

Additionally, target molecule, such as antibody etc., can be linked tothe hydrophilic polymer to targetedly deliver the conjugate of thepresent invention.

Hydrophilic polymer can be conjugated to drug molecule throughesterification reaction. This process can be simply represented asfollows:

Ester group can be eliminated by biodegradation in vivo to release theactive ingredient.

According to the present invention, the drug part of the conjugate canbe any appropriate drug molecule including, for example, amino acids,proteins, enzymes, nucleosides, saccharides, organic acids, glycosides,flavonoids, quinones, terpenoids, phenylpropanoid phenols, steroids andglycosides thereof, alkaloids and the like.

Preferably, the drug molecule included in the conjugates of the presentinvention is the active ingredient separated from nature plants, such ascinobufagin, clycyrrhetinic acid and scopoletin. Particularlypreferably, the drug molecule is the ingredient of nature medicine usedin the treatment of tumor, such as paclitaxel, camptothecin,hydroxylcamptothecin, etoposide and the derivatives thereof.

The conjugate of the present invention can be administered in the formof pure compounds or pharmaceutically acceptable compositions via anyacceptable administration route or being included in the reagent thathas the similar use. Thus, the conjugate can be administered by oral,nasal, parenteral, topical, transdermal or rectal routes in the dosageform of solid, semisolid, freeze dried powder or liquid, such astablets, suppositories, pills, soft and hard gelatin capsules, powders,solutions, suspensions and aerosols. The unit dosage forms suitable fora precise-dosage and easy administration are preferable. The compositionincludes conventional pharmaceutical carrier or excipient and theconjugate as active ingredient (one or more). The composition may alsoinclude other medical agents, carrier and adjuvant.

Generally, depending on the desired administration way, pharmaceuticallyacceptable composition includes about 1-99 wt. % of the conjugate of thepresent invention, and 99-1 wt. % of the pharmaceutically suitableexcipient. Preferably, the composition includes 5-75 wt. % of theconjugate and the rest is pharmaceutically suitable excipient.

The composition which may be administered in the form of liquid such assolution and suspension can be prepared by dissolving or dispersing theconjugate of the present invention (about 0.5-20%) and the optionalpharmaceutical adjuvant into carrier. Examples of the carrier forforming solution or suspension include water, saline, aqueous glucose,glycerol, ethanol and the like.

If needed, the composition of the present invention can further includesome additives such as wetting agent, emulsifier, pH buffer, andantioxidant etc, for example citric acid, sorbitan monolaurate,triethanolamine oleate, butylated hydroxytoluene and the like.

The practical preparation methods of such dosage forms are known orobvious to the technician in the art, for example referring toRamington's Pharmaceutical Sciences, 18^(th) edition, (Mack PublishingCompany, Easton, Pa., 1990). In any case, according to the techniques ofthe present invention, the used composition includes an effective amountof the conjugate according to the present invention to treatcorresponding diseases.

EXAMPLES

The conjugate of the present invention and its preparation method willbe further described by referring to the following examples. However,these examples do not intend to limit the scope of the invention by anymeans. The scope of the present invention is restricted only by theclaims.

Example 1

Preparation of Methoxypolyethylene glycol-glutamic Acid Oligopeptide (1)

FIG. 1 shows the synthesis method thereof. 10 g of methoxypolyethyleneglycol (molecular weight is 5000) and 2 g of N,N′-disuccinimidylcarbonate were dissolved in 100 ml of acetonitrile, and 0.5 ml of drypyridine were added thereto. Reaction mixture was stirred overnight atroom temperature under the protection of nitrogen. Excess solvents wereremoved by rotary evaporation and the residue was dried under vacuum.The obtained solid was added into 20 ml of dry dichloromethane, and themixture was filtered to remove the undissolved. The organic layer waswashed once with sodium acetate buffer solution (0.1 M, pH 5.5), driedwith anhydrous sodium sulfate, and concentrated. The product wastransferred with ether, filtered and dried under vacuum. Yield: 9.0 g(90%). NMR (DMSO): 3.5 (br m, Hs of PEG), 3.24(3H, s), 4.45(2H, t),2.82(4H, s).

0.6 g dipeptide of glutamic acid (Glu-Glu) was dissolved in 50 mlphosphate buffer solution (0.1 M, pH 7.4), and 4 g ofmethoxypolyethylene glycol succinimidyl carbonate (molecular weight5000, prepared in the former step) were added thereto. The solution wasstirred for 6 hours at room temperature and extracted 3 times withdichloromethane. The combined organic phase was dried with anhydroussodium sulfate. The solvent was removed by rotary evaporation underreduced pressure. The residue was added in 100 ml isopropyl alcohol, andfiltered. The product was dried under vacuum and purified byion-exchange chromatography. Yield: 3.6 g (90%). NMR (DMSO): 3.5 (br m,Hs in PEG), 3.24(3H, s), 4.41 (2H, t), 2.32 (4H, t).

Example 2

Preparation of Polyethylene glycol-diglutamic Acid Oligopeptide (2)

FIG. 1 shows the synthesis method thereof. 30 g of polyethylene glycol(molecular weight 35,000) and 2 g of N,N′-disuccinimidyl carbonate weredissolved in 200 ml of acetonitrile, and 0.5 ml dry pyridine was addedthereto. Reaction mixture was stirred overnight at room temperatureunder the protection of nitrogen gas. Excess solvent was removed byrotary evaporation and the residue was dried under vacuum. The obtainedsolid was added into 50 ml of dry dichloromethane and the mixture wasfiltered to remove the undissolved. The organic layer was washed oncewith sodium acetate buffer (0.1 M, pH 5.5), dried with anhydrous sodiumsulfate, and concentrated. The product was transferred by ether,filtered, and dried under vacuum. Yield: 27.2 g (90%). NMR (DMSO): 3.5(br m, Hs of PEG,), 4.45 (4H, t), 2.82 (8H, s).

0.1 g of dipeptide of glutamic acid (Glu-Glu) was dissolved in 20 ml ofdimethylformamide, and 10 g of polyethylene glycol disuccinimidylcarbonate prepared above (molecular weight 35000) were added to thesolution. Reaction mixture was stirred for 6 hours at room temperature.Solid precipitate was filtered off. The residue solution wasprecipitated with 100 ml isopropyl alcohol, filtered, and dried undervacuum. The product was purified with ion-exchange chromatography.Yield: 4.2 g (40%). NMR (DMSO): 3.5 (br m, Hs of PEG), 4.41(4H, t),2.37(4H, s), 2.32 (4H, t).

Example 3 Preparation of the Conjugate of Methoxypolyethyleneglycol-glutamic Acid Oligopeptide and Paclitaxel (3)

FIG. 2 shows the synthesis method thereof. 1.25 g of methoxypolyethyleneglycol diglutamic acid dipeptide prepared in Example 1, 0.7 g ofpaclitaxel, and 0.1 g of 4-dimethylamino pyridine (DMAP) were dissolvedinto 15 ml of dry dichloromethane, and 0.2 g of dicyclohexylcarbodiimide(DCC) was added thereto. The reaction mixture was stirred overnight atroom temperature under the protection of nitrogen gas. Excess solventwas removed by rotary evaporation and the residue was dissolved into 8ml of 1,4-dioxane. The mixture was filtered to remove the precipitateand the solution was concentrated. 30 ml of isopropyl alcohol were addedto the residue, filtered, and dried under vacuum. Yield: 1.6 g (80%).M.p.: 59˜62° C.

Example 4

The Preparation of the Conjugate of Polyethylene glycol-glutamic AcidOligopeptide and Paclitaxel (4)

4.0 g of polyethylene glycol diglutamic acid dipeptide prepared inExample 2, 0.4 g of paclitaxel and 0.08 g of 4-dimethylamino pyridine(DMAP) were dissolved into 20 ml of dry dichloromethane. After that,0.15 g of dicyclohexylcarbodiimide (DCC) were added thereto. Reactionmixture was stirred overnight at room temperature under the protectionof nitrogen gas, and the excess solvent was removed by rotaryevaporation. The residue was dissolved in 10 ml of 1,4-dioxane, filteredto remove precipitate and the mother liquid was concentrated. Theresidue was added to 50 ml of isopropyl alcohol, and filtered off. Theproduct was dried under vacuum. Yield: 3.7 g (85%). M.p.: 61-64° C.

Example 5 Preparation of the Conjugate of Methoxypolyethyleneglycol-glutamic Acid Peptide and Camptothecin (5)

FIG. 3 shows the synthesis thereof. 0.7 g of camptothecin and 0.5 g ofN-tert-butyoxylcarboxylglycine (BOC-gly) were dissolved in 10 ml of drydichloromethane. 0.62 g of dicyclohexylcarbodiimide (DCC) and 0.36 g of4-dimethylamino pyridine (DMAP) were added thereafter. The reactionmixture was stirred overnight at room temperature. The solid formedduring reaction was filtered out and the mother liquid was concentratedunder reduced pressure. The mixture was added into 50 ml ether andfiltered off. The precipitate was collected and dried under vacuum.

0.5 g of camptothecin N-tert-butyoxylcarboxylglycine ester (obtained inthe former step) was dissolved in 10 ml chloroform. Then, 10 mltrifluoroacetic acid was added. The reaction mixture was stirred for 5hours at room temperature and concentrated under reduced pressure, andthen 50 ml of diethyl ether were added. The precipitate was collected byfiltering and dried under vacuum.

2.5 g of methoxylpolyethylene glycol glutamic acid dipeptide prepared inExample 1, 0.6 g of substituted camptothecin (prepared in the formerstep), and 0.2 g of 4-dimexylamino pyridine (DMAP) were dissolved in 30ml of dry dichloromethane. 0.4 g of dicyclocarbodiimide (DCC) was thenadded. The reaction mixture was stirred overnight at room temperatureunder the protection of nitrogen gas. Excess solvent was removed byrotary evaporation, and the residue was dissolved in 15 ml of1,4-dioxane. Precipitation was filtered off and the mother liquid wasconcentrated. The residue was added to 50 ml isopropyl alcohol andfiltered off. The obtained solid was dried under vacuum. Product can bepurified by ion-exchange chromatography. Yield: 2.5 g (80%). M.p: 60-63°C.

Example 6 Preparation of the Conjugate of MethoxylpolyethyleneGlycol-Glutamic acid Oligopeptide and Cinobufagin (6)

FIG. 2 shows the synthesis thereof. 1 g of methoxylpolyethylene glycolglutamic acid dipeptide prepared in Example 1 were dissolved in 10 ml ofdichloromethane. 60 mg of cinobufagin, 32 mg of 4-dimethylamino pyridine(DMAP) and 40 mg of dicyclohexylcarbodiimide (DCC) were then added. Thereaction mixture was stirred overnight at room temperature under theprotection of nitrogen gas. The excess solvent was removed by rotaryevaporation. The residue was dissolved in 20 ml of 1,4-dioxane, filteredoff and the mother liquid was concentrated. The residue was added into100 ml isopropyl alcohol and filtered off. The obtained solid productwas dried under vacuum. Yield: 0.8 g (60%). M.p: 58˜80° C.

Example 7 Preparation of the Conjugate of Methoxylpolyethyleneglycol-glutamic Acid Oligopeptide and Clycyrrhetinic Acid

FIG. 3 shows the synthesis thereof. 1 g of methoxylpolyethylene glycolglutamic acid dipeptide prepared in Example 1 were dissolved in 10 mldichloromethane. 0.2 ml thionyl chloride was added thereto dropwise. Thereaction mixture was stirred for 2 hours. Solvent and impurities withlow boiling point were removed by distillation under reduced pressure. Asolution of 70 mg clycyrrhetinic acid in 10 ml dichloromethane was addedand dissolved by stirring. 60 mg of 4-dimethylamino pyridine (DMAP) werethen added. The reaction solution was stirred for 12 hours at roomtemperature under the protection of nitrogen gas. The solvent wasconcentrated under vacuum. The residue was added into 20 ml isopropylalcohol and filtered. The precipitate was collected, washed with diethylether, dried by suction, and further dried under vacuum. The product canbe purified by ion-exchange chromatography. Yield: 0.8 g (60%). M.p:60˜62° C.

Example 8 Preparation of the Conjugate of Methoxylpolyethyleneglycol-glutamic Acid Oligopeptide and Scopoletin (8)

FIG. 2 shows the synthesis thereof. 5 g of methoxylpolyethylene glycolglutamic acid dipeptide prepared in Example 1 were dissolved in 50 ml ofdichloromethane. 0.70 g of scopoletin, 0.1 g of 4-dimethylamino pyridine(DMAP), and 0.82 g of dicyclohexylcarbodiimide (DCC) were then added.The reaction mixture was stirred for 12 hours at room temperature underthe protection of nitrogen gas. The solvent was concentrated undervacuum. The residue was added to 20 ml of 1,4-dioxane, and filtered. Theprecipitate was collected, washed with ether and dried by suction. Themother liquid was evaporated under reduced pressure. 100 ml isopropylalcohol was added to the residue. The precipitate was collected, washedwith diethyl ether and dried by vacuum. The precipitates were combinedand dried under vacuum. Yield: 4 g (80%). M.p: 58˜61° C.

Example 9

This example is to explain the preparation process of a typicalparenteral composition. The composition comprises the conjugate of thepresent invention. Component Conjugate prepared in Example 3 2 g 0.9%saline to 100 ml

The conjugate prepared in Example 3 was dissolved in 0.9% saline toobtain 100 ml solution for intravenous injection, which was filteredthrough 0.2 μm membrane and packed sterile.

1. A conjugate of hydrophilic polymer-multicarboxyl oligopeptide anddrug molecule of the following formula:

wherein: P is a water soluble polymer; m is an integer of 2˜12; j is aninteger of 1˜6; R_(i) is a group selected from the group consisting ofH, C₁₋₁₂ alkyl, substituted aryl, aralkyl, heteroalkyl and substitutedalkyl; X is a linking group; Z is a linking group selected from O andNH; and TA is drug molecule.
 2. The conjugate of claim 1 wherein thewater soluble polymer is selected from the group consisting ofpolyethylene glycol, polypropylene glycol, polyvinyl alcohol,polyacrylmorpholine and copolymer thereof.
 3. The conjugate of claim 2wherein the water soluble polymer is polyethylene glycol.
 4. Theconjugate of claim 3 wherein the molecular weight of polyethylene glycolis 300˜60,000.
 5. The conjugate of claim 1 wherein the linking group Xis (CH₂)_(i), (CH₂)_(i)OCO, (CH₂)_(i)NHCO or (CH₂)_(i)CO, and wheren iis an integer of 0˜10.
 6. The conjugate of claim 1 wherein the freehydroxyl on the hydrophilic polymer can be substituted by C₁₋₁₂ alkoxyl,cycloalkoxyl or aroxyl.
 7. The conjugate of claim 1 wherein the freehydroxyl on the hydrophilic polymer can be substituted by the followingformula:

Wherein: x, m, j, R_(i), Z and TA are the same as defined in claim
 1. 8.The conjugate of claim 1 wherein target molecule can be carried in thehydrophilic polymer to perform targeted delivery of the conjugate. 9.The conjugate of claim 8 wherein the target molecule is an antibody. 10.The conjugate of claim 1 wherein the drug part TA is any one selectedfrom the group consisting of amino acids, proteins, enzymes,nucleosides, saccharides, organic acids, glycosides, flavonoids,quinones, terpenoids, phenylpropanoid phenols, steroids and glycosidesthereof and alkaloids.
 11. The conjugate of claim 10 wherein the drugmoiety TA is the active ingredient of a nature medicine.
 12. Theconjugate of claim 11 wherein the natural active ingredient iscinobufagin, clycyrrhetinic acid or scopoletin.
 13. The conjugate ofclaim 10 wherein the drug moiety TA is an antitumor agent.
 14. Theconjugate of claim 13 wherein the antitumor agent is selected from thegroup consisting of paclitaxel, camptothecin, hydroxylcamptothecin,etoposide and derivatives thereof.
 15. A conjugate ofmethoxylpolyethylene glycol-glutamic acid oligopeptide and drug moleculehaving the following formula:

wherein: n is an integer of 10˜1200; m is an integer of 2˜12; X is alinking group selected from the group consisting of (CH₂)_(i),(CH₂)_(i)OCO, (CH₂)_(i)NHCO and (CH₂)_(i)CO, and wherein i is an integerof 0˜10; Z is a linking group selected from O and NH; and PT is a drugselected from the group consisting of paclitaxel, camptothecin,cinobufagin, clycyrrhetinic acid, scopoletin and derivatives thereof.16. A composition comprising a conjugate according to any one of claims1 to 15 and pharmaceutically acceptable carrier or excipient.
 17. Thecomposition of claim 16 further comprising other therapeutically activeingredient.
 18. The composition of claim 16, wherein it may beformulated into the form of tablet, suppository, pill, soft and hardgelatin capsules, powder, solution, suspension, or aerosol.
 19. Use ofthe conjugate according to any one of claims 1˜15 in manufacturingmedicament.